Process for preparing 3-pyrroline-2-carboxylic acid derivatives

ABSTRACT

3-Pyrroline-2-carboxylic acid derivatives of the formula I ##STR1## are prepared by eliminating the sulfonic acid residue, with the aid of a base, from a compound of the formula II ##STR2##

This application is a 371 of PCT/EP97/03752 filed Jul. 14, 1997.

The present invention relates to a novel process for preparingpyrroline-2-carboxylic acid derivatives.

Replacement of proline by 3,4-dehydroproline in biologically activepeptides or peptide mimetics rarely causes a loss of activity (A. M.Felix et al. Int. J. Pept. Prot. Res. 10, (1977) 299; C. R. Botos et al.J. Med. Chem. 22, (1979) 926; G. H. Fisher, W. Ryan, FEBS Lett. 107,(1979) 273); on the contrary, in some cases the effect is increasedwhile there is a simultaneous reduction in toxicity (G. H. Fisher, W.Ryan FEBS Lett. 107, (1979) 273; S. Natarajan et al., in Peptide,Structure and Biological Function, E. Gross, J. Meienhofer, Eds., PierceChemical Company, 1979, p. 463).

Synthesis of N-protected 3,4-dehydroprolines on the industrial scale byprocesses disclosed in the literature is very elaborate as shown, forexample, by the thermal cis elimination of the S-methylxanthate fromhydroxyproline by the Tchugaeff method. The disadvantages of thisprocess are that large amounts of methyl iodide are used, and methylmercaptan and carbon oxysulfide are produced (J.-R. Dormay et al.,Angew. Chem. 92, (1980) 761; Houben-Weil, Methoden der OrganischenChemie, Vol. 5/1b, 126 (1972)).

The reduction of pyrrole-2-carboxylic acid with phosphonium iodide infuming hydroiodic acid is also problematic because of the use of a largeexcess of gaseous hydrogen iodide, and of a marked reduction in yieldand onset of polymerization in large reactions (J. W. Scott et al.,Synth. Commun. 10(7), (1980) 529). On the other hand, elimination of theBoc-protected 4-phenylseleninylproline methyl ester takes place underdistinctly milder conditions (J.-R. Dormay, Synthesis 9, (1982) 753. Theelimination takes place at room temperature and results in the Δ³-olefin with high selectivity. Thermal cis eliminations affordconsiderable amounts of the isomeric Δ⁴ olefin. However, elimination ofthe selenium oxide is also disadvantageous because of the production oftoxic selenium-containing residues which require costly disposalprecisely in the case of reactions on the pilot-plant scale, andaddition of the previously eliminated selenigenic acid to the doublebond is disadvantageous especially in the case of pharmaceutical activeingredients in which even tiny amounts of selenium-containing compoundsresult in toxic properties.

Small amounts of 3-pyrroline have been obtained from N-substituted3-methylsulfonyloxypyrrolidine (T. Uno et al., J. Heterocycl. Chem. 24,(1987) 1025). Elimination of sulfonates, eg. methylsulfonate, to prepare3-pyrroline-2-carboxylic acid derivatives has not previously beendescribed.

Said processes disclosed in the literature for preparing3-pyrroline-2-carboxylic acid derivatives are unsuitable for industrialsyntheses.

The present invention relates to a process for preparing3-pyrroline-2-carboxylic acid derivatives of the formula I ##STR3##where R¹ is H, C₁ -C₆ -alkyl, benzyl, benzyl substituted on the phenyl,allyloxycarbonyl, C₁ -C₆ -alkyloxycarbonyl, benzyloxycarbonyl where thebenzyl residue can be substituted by OCH₃ radicals, or C₁ -C₄-alkylcarbonyl or

R¹ is a residue of an amino acid which is linked via the C terminus andmay be alkylated or acylated on the nitrogen, and

R² is OH, C₁ -C₄ -alkyloxy, benzyloxy or NR³ R⁴,

where R³ and R⁴ are, independently of one another, H, C₁ -C₄ -alkyl,benzyl, phenyl or pyridyl, it being possible for the aromatic systems inR³ and R⁴ to be substituted by up to three identical or differentsubstituents selected from the group consisting of methyl, methoxy,hydroxyl, cyano or halogen, which comprises eliminating the sulfonicacid residue with the aid of a base from a sulfonate of the formula II##STR4## where R¹ and R² have the meanings described above, and R⁵ is C₁-C₆ -alkyl, benzyl, trifluoromethyl, naphthyl or phenyl which may beunsubstituted or substituted by radicals from the group consisting ofmethyl, nitro or halogen.

Preferred as R¹ are C₁ -C₄ -alkylcarbonyl, benzyl, benzyl substituted onthe phenyl, C₁ -C₆ -alkyloxycarbonyl and benzyloxycarbonyl. If thebenzyloxycarbonyl radical is substituted by OCH₃, it preferably has onemethoxy group in the p position. The C₁ -C₆ -alkyloxycarbonyl radical isparticularly preferred.

Preferred R² radicals are OH and C₁ -C₄ -alkoxy.

Preferred R⁵ radicals are C₁₋₆ -alkyl and benzyl, in particular C₁₋₄-alkyl.

Compounds I have one asymmetric carbon atom, and compounds II have twoasymmetric carbon atoms, in the 5-membered ring. Compounds of theformula II can be employed as racemates, mixtures of diastereomers, andas diastereomerically pure and enantiomerically pure compounds.Compounds I may therefore be obtained, depending on the stereochemicalstructure of the compounds II employed as precursors, and the reactionconditions, as racemates or in optically active form.

Compounds of the formula II can be prepared by methods disclosed in theliterature (for example D. J. Abraham, M. Mokotoff, L. Sheh, J. E.Simmons, J. Med. Chem. 26(4), (1983) 549).

Elimination of the sulfonic acid residue, ie. of the --O--SO₂ --R⁵group, from optically active compounds of the formula II takes placewith racemization if R² is C₁ -C₄ -alkoxy or benzyloxy. This results inracemic esters of 3,4-dehydroproline, which provide access, bysubsequent enzymatic racemate resolution, both to D- and toL-3,4-dehydroproline derivatives:

Process A:

(R5═CH₃, R2=OCH₃) ##STR5##

A particularly preferred embodiment of the process consists in usingcompounds of the formula II where R² is OH and the absoluteconfiguration of the carboxylic acid residue is fixed, ie. correspondseither to the R or to the S configuration, to permit the correspondingcarboxylic acids of the formula I to be obtained without racemization:

Process B:

(R5═CH₃, R2=OH) ##STR6##

Aprotic solvents are suitable for elimination reactions A and B, inparticular DMF, dioxane, THF, DME, DMSO, CH₃ CN, it being possible forthe solvent to contain small amounts of water or alcohol.

Depending on the process, 1.0-1.5 equivalents of base (process A) or2.0-3.0 equivalents of base (process B) are employed, suitable basesbeing hydrides, amides and alcoholates of lithium, sodium, potassium,rubidium, cesium, calcium or magnesium, but preferably those of sodiumand potassium. Sodium alcoholates are preferably employed as bases,suitable alcoholate residues therein being of primary, secondary andtertiary alcohols. It is also possible to employ diols, triols, etheralcohols of the tri-, di- or monoethylene glycol monoether type or aminoalcohols. Those which may be preferably mentioned are: triethyleneglycol monomethyl ether, diethylene glycol monomethyl ether or ethyleneglycol monomethyl ether, dimethylaminoethanol or2-[2-(dimethylamino)ethoxy]-ethanol.

Elimination of the sulfonate group takes place even at a temperature of-20° C. The reaction can in general be carried out from -20° C. to +100°C. It is preferably carried out at from -10° C. to 60° C. Elimination ofthe sulfonate group from the corresponding esters by process A takesplace with racemization even at -20° C. on the a carbon atom of the3,4-dehydroproline ester produced thereby.

Surprisingly, the process in the particularly preferred variant ofprocess B can be carried out with precursors of the formula II where R²is OH, and the carboxylic acid residue has either the R or Sconfiguration, almost without racemization. The bases preferablyemployed in this variant are hydrides, primary alcoholates, primaryether alcoholates or primary amino alcoholates. 2-Methoxyethanolate,2-(2-methoxyethoxy)ethanolate or 2-[2-(dimethylamino)ethoxy]ethanolateare particularly preferably employed. From 2.0 to 2.5 equivalents ofbase are preferably employed per equivalent of precursor. The preferredtemperature range for the reaction in process B is from -10° C. to +25°C.

The base used for the elimination can be introduced in solid form intothe reaction mixture, but it can also be prepared in situ before thereaction. If, for example, the base used for the elimination is sodium2-methoxyethanolate, this base can advantageously be prepared in situ bydropwise addition of the appropriate alcohol to a solution or suspensionof a sodium salt of a stronger base such as sodium hydride, sodiumtert-butoxide or sodium bis(trimethylsilyl)amide. The reaction can becarried out semibatchwise either by running the base solution into adissolved precursor of the formula II or, preferably, running a solutionof precursor II into the solution or suspension of base.

The reaction mixture can be worked up by distillation, extraction,crystallization, chromatography or a combination thereof.

The required enantiomer can be isolated from racemic 3,4-dehydroprolinewith either (+)- or (-)-tartaric acid (see J. W. Scott et al., SyntheticCommunications 10 (1980) 529 and U.S. Pat. No. 4,111,951), or theracemate resolution can be carried out with optically active1-(4-nitrophenyl)ethylamine after preparation of the Boc-protected aminoacid (U.S. Pat. No. 4,066,658, J.-U. Kahl, T. Wieland, Liebigs Ann.Chem. 8, (1981) 1445).

N-protected 3,4-dehydroprolines prepared without racemization bypreferred process B can advantageously be purified by crystallization asammonium salts with achiral amines. It is possible in particular toobtain L-boc-3,4-dehydroproline in pure form as diethylammonium salt.

The invention therefore relates to compounds of the formula IV ##STR7##where R⁶ is an amino protective group and amine is a mono-, di- ortrialkylamine where the alkyl radicals contain 1-4 carbon atoms and canbe replaced by C₅₋₇ -cycloalkyl radicals, and their optically active Dand L forms. R⁶ is preferably the boc protective group and "amine" ispreferably diethylamine or dicyclohexylamine.

Esters obtained by process A can be very effectively partially cleavedusing enzymes such as lipases, esterases and proteases, resulting in oneantipode of the free acid, while the other antipode remains in the formof the ester.

A large number of enzymes can be employed as hydrolases in the saidprocess. Proteases, esterases and, in particular, lipases are preferablyused. Microbial lipases are particularly suitable lipases and can beisolated, for example, from yeasts or bacteria. Further particularlysuitable hydrolases are the enzymes commercially obtainable from NovoNordisk (Enzyme Toolbox), in particular the lipases SP 523, SP 524; SP525, SP 526 and ®NOVOZYM 435 (Candida antarctica, lipase fraction B).

It is furthermore possible to employ the lipases "Chirazyme™ L1 to L8",which are commercially available from Boehringer Mannheim (L-1,Burkholderia cepacia, lipase; L-2, Candida antarctica, lipase fractionB; L-3, Candida rugosa, lipase; L-4, Pseudomonas sp., lipase; L-5,Candida antarctica, lipase fraction A; L-6, Psuedomonas sp., lipase;L-7, lipase, source not specified; and L-8, lipase, source notspecified), advantageously in the process according to the invention.

Esterases such as pig liver esterase can also be employed.

The enzymes can be employed in native or in immobilized form.

The ester cleavage is carried out in a buffer at pH 6-8 and preferablyat room temperature.

The novel process makes it possible to prepare compounds I in a verysimple manner. It is particularly important for preparing dehydroprolinederivatives which it has hitherto been possible to prepare only withdifficulty and, in some cases, with poor yield.

Optically active N-protected 3-pyrroline-2-carboxylic acid derivativesare obtained particularly favorably by the novel process as free acid orin the form of an ester from which the free acid can be liberated,preferably enzymatically.

If optically active acid is prepared from the ester enzymatically, as arule one antipode of the ester remains unchanged. The latter can be, forexample, racemized with bases and subjected to the enzymatic cleavageagain.

The advantage of the present invention is that it makes it possible forthe first time to prepare 3,4-dehydroproline derivatives in stericallypure form simply and under mild and, at the same time, environmentallyacceptable reaction conditions even on the industrial scale. It issurprising that elimination of the sulfonic acid residue can be carriedout even at low temperatures.

The substances prepared by the novel process are of great interest. Theyare, for example, valuable intermediates for preparing low molecularweight peptide derivatives which are thrombin inhibitors (cf. WO94/29336) and in which a proline residue is replaced by a dehydroprolineresidue. It has furthermore been found that 3,4-dehydroproline can beused to inhibit collagen synthesis (U.S. Pat. No. 4,066,658).

It is particularly advantageous for further processing that the crudeproduct obtained by the process according to the invention can bereacted without further purification to prepare the next intermediatesfor the peptide derivatives which in turn can be purified very easily.These intermediates have the formula ##STR8## where R¹ has the statedmeaning, and X is ##STR9## (R⁶ =H, CH₃, OCH₃, OH or halogen).

They can be prepared from compounds I by activating the latter in thepresence of a base such as triethylamine or diisopropylethylamine and acondensing agent such as PPA, pivaloyl chloride ordicyclohexylcarbodiimide/hydroxysuccinimide, and subsequently linkingwith H₂ N--CH₂ --X to give compounds of the formula III. The reaction isexpediently carried out in a solvent such as dichloromethane, THF,dioxane, tert-butyl methyl ether, DME or acetonitrile at from -20 to+30°.

EXAMPLES

The following abbreviations are used in the examples:

Bns=Benzylsulfonyl

Boc=tert-Butyloxycarbonyl

DIPEA=Diisopropylethylamine

DME=Dimethoxyethane

DMF=Dimethylformamide

KOtBu=Potassium tert-butoxide

Ms=Methylsulfonyl

PPA=Propylphosphonic anhydride

Pro=Proline

Pyr=3,4-Dehydroproline

RT=Room temperature

THF=Tetrahydrofuran

A. Preparation of the starting materials

a) Boc-(L)-(4-MsO)-Pro-OCH₃ and Boc-(L)-(4-BnsO)-Pro-OCH₃ :

(4R)-N-Boc-4-hydroxy-(L)-proline methyl ester was reacted withmethylsulfonyl chloride to give(4R)-N-Boc-4-methyl-sulfonyloxy-(L)-proline methyl ester (D. J. Abraham,M. Mokotoff, L. Sheh, J. E. Simmons, J. Med. Chem. 26(4), (1983) 549).Similar to the reaction with Ms chloride,(4R)-N-Boc-4-benzylsulfonyloxy-(L)-proline methyl ester is obtainedusing benzylsulfony chloride in a yield of 76% after crystallizationfrom ethanol. ¹ H-NMR (CDCl₃, δ in ppm): 7.40 and 7.28 (s, 5H,aromatic), 4.95 and 4.85 (m, 1H, O--CH), 4.40 (s, 2H, SO₂ --CH₂),4.4-4.25 (1H, N--CH), 3.72 and 3.71 (s, 3H, CO₂ CH₃), 3.7-3.50 (2H,N--CH₂), 2.53-1.95 (2H, CH₂, 1.45 and 1.42 (s, 9H, Boc); (2 rotamers).

b) (4R)-N-Boc-(4-MsO)-Pro-OH:

186 g (575 mmol) of the methyl ester Boc-(L)-(4-MsO)-Pro-OCH₃ werehydrolyzed in 500 ml of dioxane and 1150 ml of 1N NaOE at 0° C. for 2.5h. After extraction with ether, the aqueous phase was adjusted to pH 3with 2N hydrochloric acid, and the product was extracted with ethylacetate. Drying over Na₂ SO₄ and stripping off the solvent completelyresulted in 163 g (92%) of a yellowish oil which was 94% pure. Theproduct slowly solidified;

[α]_(D) ²² =-50.5° (c=1.01; MeOH); after crystallization fromdiisopropyl ether

¹ H-NMR (CDCl₃, δ in ppm): ca. 9-8 (COOH), 5.35-5.20 (m, 1H, O--CH),4.60-4.40 (1H, N--CH), 3.95-3.65 (2H, N-CH₂), 3.08 (s, 3H, SO₂ CH₃),2.85-2.25 (2H, CH₂), 1.50 and 1.40 (s, 9H, Boc); (2 rotamers)

B. Preparation of the final products

EXAMPLE 1

Preparation of Boc-(D/L)-Pyr-OCH₃ :

a) 100 g (309 mmol) of Boc-(L)-(4-MsO)-Pro-OCH₃ were dissolved in 600 mlof dry DMF. At 0-5° C., a solution of 36.45 g (325 mmol) of KOtBu in 300ml of dry DMF was added dropwise over the course of 1 h, and stirringwas continued at 0-5° C. for 30 min and at RT for 2 h. The mixture wasthen poured into ice-water and extracted three times with ether/ethylacetate 5:1, and the organic phase was again washed with water. Afterdrying over Na₂ SO₄, the solvent was completely stripped off at 35° C.68 g of crude ester were obtained and were distilled at 100-102° C.under 1.7 mbar. The resulting colorless oil solidified (56% yield).

¹ H-NMR (CDCl₃, δ in ppm): 6.05-5.95 (m, 1H, --CH═CH--), 5.80-5.67 (m,1H, --CH═CH--), 5.05 and 4.98 (m, 1H, N--CH), 4.35-4.15 (m, 2H, N--CH₂),3.75 and 3.74 (s, 3H, CO₂ CH₃), 1.47 and 1.43 (s, 9H, Boc); (2 rotamers)

b) The same compound (Boc-(D/L)-Pyr-OCH₃) was also obtained from thebenzylsulfonate Boc-(L)-(4-BnsO)-Pro-OCH₃. From 5 g (12.5 mmol) of saidbenzylsulfonate in 50 ml of dry DMF, which was added dropwise to asuspension of 0.5 g (12.5 mmol) of NaH in 10 ml of dry DMF at -10° C.and then stirred at RT overnight, there were obtained after workupsimilar to the elimination of the mesylate in a) 2.2 g of crude ester.The product was purified by column chromatography on silica gel (mobilephase: ethyl acetate/hexane 2:3). However, distillation is preferablebecause the chromophore is weak.

EXAMPLE 2

Preparation of Boc-(L)-Pyr-OH:

a) 50.0 g (161.6 mmol) of Boc-(L)-(4-MsO)-Pro-OH dissolved in 650 ml ofDME (to which 25 mmol of water were added) were added dropwise in 45 minto 14.5 g of 55-65% NaH (about 364 mmol) in 400 ml of DME at roomtemperature, the temperature rising without additional cooling to about30° C. The mixture was stirred at RT for a further 15 h and then at 50°C. for 1 h, subsequently poured into ice-water and washed three timeswith ether/ethyl acetate 2:1. The aqueous phase was acidified to pH 2with 2N hydrochloric acid, and the product was extracted with ethylacetate. Drying over Na₂ SO₄ and stripping off the solvent completelyresulted in 36 g of crude substance as yellowish oil which contained 70%of product. The product/precursor ratio was found to be 97:3 (HPLC:water/acetonitrile 8:2+0.1% TFA; Merck ®PUROSPHER RP-18e; detection at210.4 nm) and the enantiomer ratio (L):(D) was 90:10. The proportions ofenantiomers were detected on a chiral HPLC column asBoc-3,4-dehydroprolyl 3-picolylamide after coupling the acidic groupwith a 3-picolylamine derivative. Previous investigations have shownthat the coupling itself takes place virtually without racemization.

A similar reaction but with 3 equivalents of NaH and stirring at RT for4 h resulted in 65% of product (product:precursor=94:6; (L):(D)=96:4).

¹ H-NMR (CDCl₃, δ in ppm) : 10.5-9.5 (COOH), 6.10-5.90 (1H, --CH═CH--),5.88-5.70 (1H, --CH═CH--), 5.12-4.95 (1H, N--CH), 4.30-4.15 (2H, N=13CH₂), 1.55-1.35 (9H, Boc); (2 rotamers)

By classical racemate resolution methods, Boc-3,4-dehydroproline and(+)-dehydroabietylamine were crystallized as corresponding ammonium saltfrom acetone and, without further recrystallization and afterelimination of the amine, Boc-(L)-3,4-dehydroproline was isolated with apurity of 85% and an enantiomer ratio (L):(D) 96:4.

b) 46.2 g of sodium tert-pentoxide (398.5 mmol) were introduced into 150ml of THF. Then, at 10° C., 32.9 g of 2-methoxyethanol (429.5 mmol) wereadded. A solution of 50 g of Boc-(L)-(4-MsO)-Pro-OH (159.4 mmol) in 100ml of THF was then added dropwise in such a way that the internaltemperature did not exceed 8-10° C. The mixture was stirred at 10° C.for a further 20 h after the end of the addition. Addition of 300 ml ofice-water at 5-10° C. was followed by one extraction with 50 ml ofmethyl tert-butyl ether and then acidification to pH 2 with hydrochloricacid. The crude product was extracted with methylene chloride and, afterthe solvent had been evaporated off, isolated as yellow oil. The weightwas 40.9 g, of which 18 g was Boc-(L)-3,4-dehydroproline, determined byHPLC analysis calibrated with external standard (initial gradient water(0.1% H₃ PO₄)/acetonitrile 70:30; column: Prodigy (ODS3) 100A; detectionat 210 nm). The (L):(D) enantiomer ratio of 99:1 was likewise determinedby HPLC analysis (hexane/isopropanol 8.75:1.25, 0.1% HCOOH; column:Chiracel OD; detection at 230 nm).

70 g of Boc-(L)-(4-MsO)-Pro-OH (224 mmol) were reacted under similarconditions. After extraction with methylene chloride, the crude productwas transferred into 220 ml of methyl tert-butyl ether by distillativesolvent exchange, and then the Boc-(L)-3,4-dehydroproline presenttherein was precipitated as diethylammonium salt by adding 16.5 g ofdiethylamine (224 mmol). 23.8 g of this salt were obtained. The (D)enantiomer was undetectable by the HPLC analytical methods indicatedabove in the product precipitated in this way.

¹ H-NMR (DMSO, δ in ppm): 5.86-5.67 (2H, --CH═CH--), 4.6-4.5 (N--CH),4.1-3.9 (N--CH₂), 2.88-2.7 (4H q, NCH₂ CH₃), 1.45-1.25 (9H, Boc 2rotamers), 1.2-1.05 (6H, NCH₂ CH₃) [α]_(D) ²² =-240.1° (C=1.08, MeOH)Melting point: 130-133° C.

c) 10.52 g of sodium bis(trimethylsilyl)amide (57.4 mmol) wereintroduced into 25 ml of THF and, after dropwise addition of 8.25 g of2-[2-(dimethylamino)ethoxy]ethanol (62 mmol) in 15 ml of THF over thecourse of 15 min with cooling, stirred at RT for 30 min. Then, at -5°C., 7.1 g of Boc-(L)-(4-MsO)-Pro-OH (23.0 mmol) dissolved in 15 ml ofTHF were added dropwise over the course of 20 min, and the mixture wasstirred at -5° C. for 1 h, at 0° C. for 2 h and at RT overnight. It wasthen poured onto 125 g of ice-water and extracted four times with methyltert-butyl ether, and the aqueous phase was acidified to pH 2.2 with 60ml of 10% strength citric acid and stirred at RT overnight. After thereaction solution had been extracted with methyl tert-butyl ether threetimes, the collected organic phases were washed successively with water,saturated brine and water, dried over magnesium sulfate and concentratedunder reduced pressure. 4.1 g of Boc-(L)-3,4-dehydroproline wereobtained as crude product which was then dissolved in 20 ml of methyltert-butyl ether, and a solution of 1.35 g of diethylamine (18.52 mmol)in 10 ml of methyl tert-butyl ether was added dropwise. Petroleum etherwas added to complete precipitation of the salt. The product wasfiltered off with suction and dried to afford 4.0 g ofBoc-(L)-3,4-dehydroproline. A second batch of 0.3 g of crystals was alsoobtained from the mother liquor, which means that the total yield ofrequired product was 66%.

EXAMPLE 3

Preparation of Boc-(D,L)-Pyr-OH:

13 g of isopropanol (215 mmol) were added dropwise to 8 g of 60% NaH(200 mmol) in 150 ml of DME with cooling. After evolution of H₂ hadsubsided, at 0° C. a solution of 25 g of Boc-(L)-(4-MsO)-Pro-OH (80mmol) in 100 ml of DME was added. 1 h at 0° C. was followed by warmingat 20° C. for 20 h, and then addition of 150 ml of water. One extractionwith methyl tert-butyl ether was followed by acidification to pH 2 withhydrochloric acid and extraction with methylene chloride. The weight ofproduct was 17 g. The (L):(D) enantiomer ratio was determined by HPLCanalysis (hexane/isopropanol 8.75:1.25, 0.1% HCOOR; column: Chiracel OD;detection at 230 nm) as 57:43.

EXAMPLE 4

Enzymatic cleavage of Boc-(D/L)-Pyr-OCH₃ to Boc-(L)-Pyr-OH:

5.68 g (25 mmol) of Boc-(D/L)-Pyr-OCH₃ in 100 ml of phosphate buffer (pH7.0) and 15 ml of THF were shaken with 3.12 g of ®NOVOZYM 435 at RT for24 h. During this, the pH was adjusted back to the initial value byadding 1N NaOH. The progress of the reaction was observed from theconsumption of sodium hydroxide solution. The solid was filtered off,and the filtrate was adjusted to pH 10 with 1N NaOH. The unreactedanti-pode Boc-(D)-Pyr-OCH₃ was extracted with ethyl acetate/ether 1:1.The aqueous phase was adjusted to pH 1 with 1N hydrochloric acid, andthe product Boc-(L)-Pyr-OH was extracted three times with ethyl acetate.2.14 g of product were obtained and were crystallized fromtoluene/hexane or ether/hexane. After crystallization, the product hadan optical rotation of [α]_(D) ²² =-273.8° (c=1.03; methanol). (Lit.:[α]_(D) ²⁵ =-272° (c=1.0; methanol) J.-U. Kahl, T. Wieland, Liebigs Ann.Chem. 8, (1981) 1445).

USE EXAMPLE 1

Preparation of H-(L)-Pyr (6-carboxamido)-3-picolylamide dihydrochloride:

5.3 ml (30.3 mmol) of DIPEA were added dropwise to 1.5 g of crudeBoc-Pyr-OH from Example 2 in 30 ml of dichloromethane at -10° C. and,after 5 min, 1.58 g (7.0 mmol) of (6-carboxamido)-3-picolylamidedihydrochloride were added and, after a further 5 min, 5.7 ml (7.9 mmol)of PPA (50% strength solution in ethyl acetate) in 5 ml ofdichloromethane were added. The reaction mixture was allowed to warmfrom -10° C. to 0° C. over 1 h and was then diluted with dichloromethaneand washed successively with saturated NaHCO₃ solution, 5% strengthcitric acid and saturated brine. Drying of the organic phase over Na₂SO₄ and stripping off the solvent completely resulted in 1.8 g of crudeBoc-(L)-Pyr (6-carboxamido)-3-picolylamide which was stirred in 30 ml of0.9 molar isopropanolic HCL at 50° C. for 50 min. The precipitate whichwas produced during this was removed on a suction filter, dissolved in alittle methanol, precipitated with isopropanol and again removed. Dryingat 45° C. under reduced pressure resulted in 2.0 g of H-(L)-Pyr(6-carboxamido)-3-picolylamide dihydrochloride as white powder (purity95%); (L):(D)>99:1.

¹ H-NMR (DMSO-d⁶, δ ppm) : 10.9 and 8.9 (each 1H, --NH₂ --⊕), 9.77 (t,1H, CO--NH), 8.60, 8.10 and 8.00 (each 1H, aromatic H), 8.25 and 7.75(each 1H, CO--NH₂), 6.03 (s, 2H, --CH═CH--), 5.10 (1H, N--CH--CO) 4.47(d, 2H, CH₂) 4.00 (2H, CH₂)

USE EXAMPLE 2

Preparation of H-(L)-Pyr 4-CN-benzylamide hydrochloride:

10.0 g of crude Boc-Pyr-OH were reacted with 6.2 g of p-cyanobenzylamineas in Use Example 1. Workup resulted in 14.7 g of crude Boc-(L)-Pyr4-CN-benzylamide which was stirred in 230 ml of 1 molar isopropanolicHCl at 50° C. for 2 h. After the solution had cooled to RT, thesubstance began slowly to precipitate. The solid was removed on asuction filter. 3.7 g of H-(L)-Pyr 4-CN-benzylamide hydrochloride wereobtained as a white powder (purity 96%; (L):(D)>99:1). ¹ H-NMR (DMSO-d⁶,δ in ppm) : 10.9 and 8.9 (each 1H, --NH₂ --⊕), 7.82 and 7.47 (each 2H,aromatic H), 6.02 (s, 2H, --CH═CH--), 5.10 (1H, N--CH--CO) 4.45 (d, 2H,CH₂) 4.02 (2H, CH₂)

We claim:
 1. A process for preparing 3-pyrroline-2-carboxylic acidderivatives of the formula I ##STR10## where R¹ is H, C₁ -C₆ -alkyl,benzyl, benzyl substituted on the phenyl, allyloxycarbonyl, C₁ -C₆-alkyloxycarbonyl, benzyloxycarbonyl where the benzyl residue can besubstituted by OCH₃ radicals, or C₁ -C₆ -alkylcarbonyl orR¹ is a residueof an amino acid which is linked via the C terminus and may be alkylatedor acylated on the nitrogen, and R² is OH, C₁ -C₆ -alkyloxy, benzyloxyor N R³ 4², where R³ and R⁴ are independently of one another, H, C₁ -C₆-alkyl, benzyl, phenyl or pyridyl, the aromatic systems in R³ and r⁴being optionally substituted by up to three identical or differentsubstituents selected from the group consisting of methyl, methoxy,hydroxyl, cyano or halogen, which comprises eliminating the sulfonicacid radical with the aid of a base selected from the group consistingof hydrides, amides and alcoholates of lithium, sodium, potassium,rubidium, cesium, calcium or magnesium from a compound of the formula II##STR11## where R¹ and R² have the meanings described above, and R⁵ isC₁ -C₆ -alkyl, benzyl, trifluoromethyl, naphthyl or phenyl which may beunsubstituted or substituted by radicals from the group consisting ofmethyl, nitro or halogen.
 2. A process as claimed in claim 1 forpreparing an optically active 3-pyrroline-2-carboxylic acid, wherein thesulfonic acid radical is eliminated with the aid of a base selected fromthe group consisting of hydrides, amides and alcoholates of lithium,sodium, potassium, rubidium, cesium, calcium or magnesium from acompound of the formula II, where R¹ and R⁵ have the meanings describedabove, and R² is C₁ -C₄ -alkyloxy or benzyloxy, and the optically activefree acids are liberated enzymatically from the obtained racemic esterin the form of ammonium salts.
 3. A process as claimed in claim 1 forpreparing an optically active 3-pyrroline-2-carboxylic acid, wherein thesulfonic acid radical is eliminated with the aid of a base selected fromthe group consisting of hydrides, amides and alcoholates of lithium,sodium, potassium, rubidium, cesium, calcium or magnesium from acompound of the formula II where the carboxylic acid residue has eitherthe (R) or the (S) configuration, R¹ and R⁵ have the abovementionedmeanings, and R² is OH, and the obtained products are optionallyconverted into ammonium salts for purification and isolation.
 4. Aprocess as claimed in claim 1 for preparing 3-pyrroline-2-carboxylicacid derivatives and subsequently converting the obtained3-pyrroline-2-carboxylic acid derivatives to compounds of the formula II##STR12## where R¹ has the stated meaning, X is ##STR13## R⁶ is H, CH3,OCH3, OH or halogen.
 5. A compound of the formula IV ##STR14## where R¹is an amino protective group selected from allyloxycarbonyl, C₁ -C₆-alkyloxycarbonyl, benzyloxycarbonyl or C₁ -C₄ -alkylcarbonyl, and amineis a mono-, di- or trialkylamine where the alkyl radicals contain 1-4carbon atoms and may be replaced by C₅₋₇ -cycloalkyl radicals.
 6. Acompound of the formula IV as claimed in claim 5, where R¹ is the Bocprotective group and the amine is diethylamine or dicyclohexylamine. 7.A compound as claimed in claim 5 in the D form.
 8. A compound as claimedin claim 5 in the L form.